1. Field of the Invention
The present invention relates to cellular screening substrata capable of being used for identifying substances contributing to at least one of cell adhesion, proliferation, differentiation, survival, maintenance of undifferentiated state, and apoptosis, to their manufacturing processes, and to methods and apparatus for cellular screening using the cellular screening substrata.
2. Related Background Art
Recently, there have been actively carried out studies on culture of animal and plant cells under different conditions, or studies on products of particular cultured cells. Specifically, investigations have been made in various fields to produce, utilizing specific cellular activities, substances which are impossible to synthesize artificially or otherwise quite difficult to synthesize.
Also, there have been carried out studies to identify substances having an effect on cell proliferation and differentiation to proliferate or differentiate desired cells according to intended applications. With rapid progress in cell technology and medical engineering, attention has focused on microbiosensors or artificial organs using cells, and furthermore on neurocomputers and the like, and active research efforts have been made for these applications.
In order to utilize cells in vitro as stated above, however, it is indispensable to position cells as desired and to control their proliferation, differentiation, and production of substances. Mechanisms that control cell positioning, proliferation, differentiation, and production of substances, however, have not been sufficiently elucidated yet. Thus, it is quite difficult to culture cells controlling these cellular functions, which is one of the main obstacles to researches and developments utilizing cells as described above.
As an attempt to regulate the cell positioning, U.S. Pat. No. 5,108,926 discloses a method employing an ink jet printer to form a pattern of a cell adhesive protein on which cells are grown. By this method, cells can be cultured on the surface of the pattern where a cell adhesive protein was applied, but it is impossible to control their proliferation/differentiation and production of substances to screen the cells.
In an article (Proteins, Nucleic Acids and Enzymes, 45-5, 727-734 (2000), cell growth factors that effect proliferation and differentiation of cells were immobilized onto a support using a photolithography technique, and their effects on proliferation and differentiation of cells were studied. The substrate on which the cell growth factors had been immobilized, however, was not used as means for cellular screening. In addition, photolithographic procedures waste biological substances that exist only in small amounts in the body, and necessitate repeating the processes of exposure and development, complicating the production steps.
WO97/45730 proposes a method of screening cells by immobilizing a substance that influences cell adhesiveness onto a substrate. In this method, a reactive functional group provided on the substratum and a cell adhesive material is immobilized through a divalent crosslinking reagent. This method employs photolithography to bind the reactive functional group to the cell adhesive material. Thus, this method has problems as described above. In addition, when two or more cell adhesive materials are immobilized, it is almost impossible to avoid the binding of the material which has been already immobilized and a material to be newly immobilized at undesired locations through a divalent crosslinking reagent, and therefore it is very difficult to position cell adhesive materials on desired locations. Further, this method does not include immobilization of substances that affect cell proliferation, differentiation or production. In addition, according to this method, cells are fixed in wells through the cell adhesive materials, and screening is carried out by detecting substances produced by the cells during culture of the cells. Thus, this method is not a method for screening substances which have an effect on proliferation and differentiation, and furthermore production of substances, as in the present invention.